1. Field of the Invention
This invention is in the field of recombinant protein expression. In particular, the invention relates to techniques for the rapid screening of suitable translational fusion partners (TFPs) capable of inducing secretory production of recombinant proteins, especially proteins that are difficult to produce using conventional recombinant production methods.
2. Related Art
The recombinant expression of proteins of interest is a widely used; procedure to produce large quantities of proteins for research purposes or for therapeutic and other commercial uses. A variety of recombinant expression systems are known in the art, including bacterial, yeast, and mammalian host cell systems, and many different proteins have been successfully produced in these systems. However, there are also many proteins that are not easily produced using available expression systems, resulting in little or no protein expression and secretion. Methods for improving the secretion of recombinantly expressed proteins, such as overexpressing secretory factors in the host cells, using fusion proteins comprising the protein of interest fused to a well-secreted protein, and adding synthetic linker sequences, have had some success with particular proteins of interest. However, no general technique has been identified that is effective for the secretory production of all proteins.
In an effort to identify secreted proteins and novel signal sequences, several signal sequence trap systems have been developed. U.S. Pat. No. 6,228,590 describes a technique for screening for mammalian signal sequences by transforming reporter protein-deficient yeast with nucleic acids comprising mammalian coding sequences fused to a reporter protein and detecting cells that secrete the reporter protein. A similar system using invertase-deficient yeast and an invertase reporter protein is disclosed in EP0907727. Yeast-based signal sequence traps have been used to identify secreted proteins from human DNA Klein et al., Proc. Natl. Acad. Sci. USA 93:7108 (1996); Jacobs et al., Gene 198:289 (1997)), mouse DNA (Gallicioti et al., J. Membrane Biol. 183:175 (2001)), zebrafish DNA (Crosier et al, Dev. Dynamics 222:637 (2001)), Arabidopsis DNA (Goo et al., Plant Mol. Biol. 41:415 (1999)), potato DNA (Surpili et al., Anais de Academia Brasileira de Ciencias 74:599 (2002)), and Candida albicans DNA (Monteoliva et al., Eukaiyotic Cell 1:514 (2002)). Similar trap systems have been developed using mammalian host cells (Gallicioti et al., J. Membrane Biol. 183:175 (2001)) and bacterial host cells (Ferguson et al., Cancer Res. 65:8209 (2000). Reporter proteins that have been used in signal sequence traps include invertase (Klein et al., Proc. Natl. Acad. Sci. USA 93:7108 (1996)), alpha amylase (U.S. Pat. No. 6,228,590), acid phosphatase (PHQ5) (Surpili et al., Anais de Academia Brasileira de Ciencias 74:599 (2002)), and β-lactamase Ferguson et al., Cancer Res. 65:8209 (2000).
A method for identifying translational fusion partners (TFPs) useful for secretion of a target protein is disclosed in WO 2005/068658. The method comprises (i) obtaining a plurality of host cells transformed with a variety of vectors comprising a library of nucleic acid fragments and a target protein-encoding nucleotide sequence fused with a reporter protein-encoding nucleotide sequence, wherein the host cells are deficient in the reporter protein, and (ii) identifying a TFP library from the host cells, wherein the TFP library comprises nucleic acid fragments which individually induce the secretion of the target protein.